UV mutagenesis and tolerance adaptive laboratory evolution of Pichia fermentans modulate the membrane- bound xylose uptake transporters (XUT) genes for enhanced xylitol production

UV mutagenesis and tolerance adaptive laboratory evolution of Pichia fermentans modulate the membrane- bound xylose uptake transporters (XUT) genes for enhanced xylitol production | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article UV mutagenesis and tolerance adaptive laboratory evolution of Pichia fermentans modulate the membrane- bound xylose uptake transporters (XUT) genes for enhanced xylitol production Ramalingam Kayalvizhi, Samuel Jacob This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8818678/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Development of a strain improvement strategy is inevitable for the industrial production of commercial chemicals. In this study, a promising yeast, Pichia fermentans NCIM 3638 was selected for metabolic modulation aimed at xylitol (a low-calorie sweetener) production. The strain was subjected to UV mutagenesis followed by sequential LiCl-induced oxidative stress to modulate xylose metabolism for enhanced xylitol production. The resultant mutant strain, P. fermentans KS-MUT9, achieved a maximum xylitol yield of 0.61 g/g xylose, representing a 1.61-fold increase compared to the wild type. Analysis of key enzymes involved in xylose metabolism revealed a 7.47-fold increase in xylose reductase activity (1.27 IU/mg) and a 0.22-fold decrease in xylitol dehydrogenase activity (0.11 IU/mg) in the mutant strain relative to the wild-type, correlating with the enhanced xylitol yield. Molecular investigations using qPCR demonstrated upregulation of the xylose reductase gene (XYL1, 3.89-fold), xylitol dehydrogenase gene (XYL2, 1.91-fold), and a substantial 14.93-fold increase in the xylose uptake transporter gene-4 (XUT4), supporting metabolic rewiring through the adopted strain improvement strategy. Additionally, Sanger sequencing identified six and four nucleotide substitutions in XUT6 and XUT7 of KS-MUT9, respectively. Furthermore, to assess industrial scalability, a mathematical evaluation of the fermentative potential of the mutant strain was conducted to determine critical scale-up kinetic parameters (Xc, Sc, Pc) using unstructured kinetic modeling. The mutant strain developed through UV mutagenesis and LiCl-assisted tolerance adaptive laboratory evolution exhibited a reprogrammed metabolic profile favoring enhanced xylitol production, highlighting its potential for industrial bioproduction without ethical or regulatory concerns. Pichia fermentans Strain improvement Tolerance adaptive laboratory evolution UV mutagenesis Xylitol Xylose reductase Xylose uptake transporters Full Text Additional Declarations No competing interests reported. Supplementary Files Supplementaryinformation.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8818678","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":591536287,"identity":"4fc93f1a-b2b8-4fc4-a112-d95f51a6866a","order_by":0,"name":"Ramalingam Kayalvizhi","email":"","orcid":"","institution":"SRM Institute of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Ramalingam","middleName":"","lastName":"Kayalvizhi","suffix":""},{"id":591536288,"identity":"c8ba7f81-9f2b-4b8c-84a5-74e3f252785a","order_by":1,"name":"Samuel Jacob","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA8UlEQVRIiWNgGAWjYNCCArYEfhCdwAAlEwhqMWBLkGwAKzYgWgtDgsEBKIugevn202kSHwz48ozPn3264cGfPwz87DkGDA934DH/TO42yRkGbMVmN9LNbiS2GTBI9rwxYEg8g89JudukeQzYErfdYGO7kdhgwGBwA2hLYhseh/W/3Sb9B6hlc/8xthsJfwwY7AlpYbgBtAUYYokbGNKAWtiAtkgQ0GJw4+1myx6gXyRuALUkthnzSJx5VnAAv8NyN974UXEsjx/osJs//sjJ8bcnb3z4E5/DIOAYnMUDIg4Q1MDAUEOEmlEwCkbBKBixAABZYlH4fGDh1QAAAABJRU5ErkJggg==","orcid":"","institution":"SRM Institute of Science and Technology","correspondingAuthor":true,"prefix":"","firstName":"Samuel","middleName":"","lastName":"Jacob","suffix":""}],"badges":[],"createdAt":"2026-02-08 02:23:34","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8818678/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8818678/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":102964481,"identity":"e025f5e2-3aad-499d-9ae0-03bc2c1c067d","added_by":"auto","created_at":"2026-02-19 04:22:26","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1875253,"visible":true,"origin":"","legend":"","description":"","filename":"Manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8818678/v1_covered_3faf7b62-6f80-4083-934c-79a1bf361737.pdf"},{"id":102918070,"identity":"b302f5c2-7ce0-4fb9-ac33-dfaed60af369","added_by":"auto","created_at":"2026-02-18 11:47:36","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":2273115,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryinformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-8818678/v1/3339be8985ef01e6f003d121.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"UV mutagenesis and tolerance adaptive laboratory evolution of Pichia fermentans modulate the membrane- bound xylose uptake transporters (XUT) genes for enhanced xylitol production","fulltext":[],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":true,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":true,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Pichia fermentans, Strain improvement, Tolerance adaptive laboratory evolution, UV mutagenesis, Xylitol, Xylose reductase, Xylose uptake transporters","lastPublishedDoi":"10.21203/rs.3.rs-8818678/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8818678/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Development of a strain improvement strategy is inevitable for the industrial production of commercial chemicals. In this study, a promising yeast, Pichia fermentans NCIM 3638 was selected for metabolic modulation aimed at xylitol (a low-calorie sweetener) production. The strain was subjected to UV mutagenesis followed by sequential LiCl-induced oxidative stress to modulate xylose metabolism for enhanced xylitol production. The resultant mutant strain, P. fermentans KS-MUT9, achieved a maximum xylitol yield of 0.61 g/g xylose, representing a 1.61-fold increase compared to the wild type. Analysis of key enzymes involved in xylose metabolism revealed a 7.47-fold increase in xylose reductase activity (1.27 IU/mg) and a 0.22-fold decrease in xylitol dehydrogenase activity (0.11 IU/mg) in the mutant strain relative to the wild-type, correlating with the enhanced xylitol yield. Molecular investigations using qPCR demonstrated upregulation of the xylose reductase gene (XYL1, 3.89-fold), xylitol dehydrogenase gene (XYL2, 1.91-fold), and a substantial 14.93-fold increase in the xylose uptake transporter gene-4 (XUT4), supporting metabolic rewiring through the adopted strain improvement strategy. Additionally, Sanger sequencing identified six and four nucleotide substitutions in XUT6 and XUT7 of KS-MUT9, respectively. Furthermore, to assess industrial scalability, a mathematical evaluation of the fermentative potential of the mutant strain was conducted to determine critical scale-up kinetic parameters (Xc, Sc, Pc) using unstructured kinetic modeling. 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